1. Field of the Invention
This invention involves a method and an apparatus for optically detecting trace chemicals near a sensor surface.
2. Description of the Related Art
Many branches of medicine, chemistry, and biology depend on an ability to assay chemical, biochemical, or biological samples, or to determine changes in the chemical composition of such samples. The diagnosis of many diseases, for example, often relies on the ability to detect the presence of antibodies in the blood.
As another example, using fluorometric analysis, one determines the amount of a fluorescing material present in a sample by measuring the intensity of the fluorescence emitted from the material. Such fluorometric techniques are particularly useful in biochemical assays that test for the presence of fluorophores.
There are accordingly many different methods and devices for detecting the presence or measuring the amount of chemicals in a bulk sample. These devices are well understood and have been widely used. Examples of these devices are described in the books Laser Spectroscopy, Demtroder, W., Springer-Verlag, Berlin, 1982, and Fluorescence and Phosphorescence, Rendell, D., John Wiley, Chichester, 1987.
Other known chemical sensors involve optical absorption spectroscopy. These spectroscopic analyzers typically use one of two techniques: 1) sensing of interferences between multiple reflections in thin films, as is described in Internal Reflection Spectroscopy, Harrick, N.J., Wiley, New York, 1967; and 2) total internal reflection (TIR) absorption spectroscopy inside a laser cavity, as is described in "Intracavity adsorption spectroscopy of surface-active substances, adsorbed gases, and aerosols," Godlevskii, A.P., and Kopytin, Yu. D., J. Appl. Spect., 29, 1301 (1978).
Harrick describes the use of constructive interferences in thin film "cavities" to increase the sensitivity of optical absorption spectroscopy. Light is coupled into a thin film where it undergoes multiple (total internal) reflections. If a wide light beam is used, then multiply reflected beams may constructively interfere with the directly reflected beam. Harrick points out, however, that many problems exist for this structure: precise angles of incidence need to be maintained, effective improvement is limited by the actual size of the beam, and critical matching of surface reflectivities is essential for optimum enhancement.
Recent work has shown that the latter limitation can be relieved. U.S. Pat. No. 4,857,273 (Stewart, 15 Aug. 1989, "Biosensors"), for example, illustrates improvements over the Harrick system. Other problems, however, so limit the sensitivity of this structure that they render it practically useless as a highly sensitive transducer for small sensing areas. Many of these problems stem from the fact that the Stewart sensor employs a form of resonant waveguide and thus requires a long interaction length so that light will "bounce" enough times within the waveguide. To make the device smaller, one would have to shorten the interaction length. This would, though, also reduce the length over which light could bounce and would cause the sensor to lose much of its sensitivity.
Godlevskii and Kopytin describe a system in which a total reflecting cell is placed inside a laser resonator. The output laser power is then measured as molecules are adsorbed to the TIR surface. The molecules are assumed to have an optical absorption at or near the laser frequency, which means that they will act as an additional intracavity loss mechanism. While both the Harrick and Godlevskii systems use "optical cavities," only the latter offers an optical resonator that is stable enough to solve many of Harrick's problems.
On the other hand, the Godlevskii/Kopytin system confines itself to absorption spectroscopy of simple molecules. In order for the Godlevskii/Kopytin system to work properly, it must therefore have enough molecules of the analyte to provide detectable absorption. Because of this, the sensitivity of the Godlevskii/Kopytin system is limited to detection of surface concentrations greater than those at which the intracavity field is significantly perturbed. Since this system relies on having enough absorbing molecules of the analyte to perturb the electric field at the TIR boundary, the system is not able to detect concentrations or molecules much below this limit.
According to another known technique, trace chemicals are detected using fluorescence. A method for detecting biomolecules using fluorescence at a TIR surface is, for example, described in the article "A new immunoassay based on fluorescence excitation by internal reflection spectro-scopy," by Kronick, M. L., and Little, W. A., J. Immunological. Meth., 8, 235 (1975). According to this technique, a prism is mounted in free space, not within a resonant cavity. Because of this structure, light is deliberately "discarded," that is, its energy is not used to contribute further to fluorescence, after it is reflected off of the TIR surface. This in turn means that the Kronick system requires large and powerful light sources in order to achieve sufficient fluorescent excitation for high sensitivity.
Other detection systems and techniques that use fluorescent excitation are described in the following references:
1) Sloper, A. N., Deacon, J. K., and Flanagan, M. T., "A planar indium phosphate monomode waveguide evanescent field immunosensor," Sensors and Actuators, B1, 589 (1990), (describing the use of waveguides); PA1 2) Choquette, S. J., Locascio-Brown, L., and Durst, R. A., "Planar waveguide immunosensor with fluorescent liposome amplification," Anal. Chem., 64, 55 (1992), (also using waveguides); and PA1 3) Kooyman, R. P. H., de Bruijn, H. E., and Greve, J., "A fiber-optic fluorescence immunosensor," Proc. Soc. Photo-Opt. Instrum. Eng., 798, 290 (1987), (describing the use of optic fibers).
As in the Kronick method, these three techniques do not "recycle" light within a cavity, and are thus similarly limited either to needlessly large light sources or to reduced sensitivity.
By definition, the less of a material a detector needs in order to detect it, the more sensitive the detector will be. Consequently, it is a standing goal to increase the sensitivity of detectors.
There is a well-recognized need for techniques that an rapidly detect minute amounts (for example, fewer than 10.sup.5 molecules), of biomolecules without either radioactive labeling or chemical amplification (such as polymerase chain reaction). Single molecules have been detected optically using methods described in Peck, K., Stryer, L., Glazer, A. N., and Mathies, R. A., "Single molecule fluorescence detection: Autocorrelation criterion and experimental realization with phycoerythrin," Proc. Natl. Acad. Sci. USA, 86, 4087 (1989), and Soper, S. A., Brooks Shera, E., Martin, J. C., Jett, J. H., Hahn, J. H., Nutter, H. L., and Keller, R. A., "Single-molecule detection of rhodamine 6G in ethanolic solutions using continuous wave laser excitation," Anal. Chem., 63, 432 (1991). The problem with these methods, however, is that they are very difficult to produce commercially, since they, too, require large lasers (with accompanying large power supplies and in some cases cooling systems), to generate a sufficiently strong electric field.
Apart from the question of sensitivity, many existing detection systems also suffer from the problem that they are bulky or are difficult to control electronically. This means not only that they are less accessible to small laboratories, which may have neither the space nor the money to buy and install one, but also that they will be harder to use and control properly if they are installed.
What is needed is a method for surface (rather than only bulk), detection that makes it possible to detect sample concentrations of molecules even less that those for which the intracavity field is perturbed. The detection system itself should be stable, compact, easily calibrated, and easily controlled.